Development of a 16S rRNA Gene Primer and PCR-Restriction Fragment Length Polymorphism Method for Rapid Detection of Members of the Genus Megasphaera and Species-Level Identification

Ohnishi, Akihiro; Abe, Shinko; Nashirozawa, Shiho; Shimada, Sawahiko; Fujimoto, Naoshi; Suzuki, Masaharu

doi:10.1128/aem.00359-11

Cited by 15 publications

(9 citation statements)

References 11 publications

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“…The universal primer set 20F/1540R was used as the positive control (Ohnishi et al, 2011b). The analytical specificity of the constructed primer set Mm2F/Mega-X was evaluated using PCR.…”

Section: Resultsmentioning

confidence: 99%

“…All strains for this study (Table 1) were obtained from a culture collection of prokaryotes and handled in an anaerobic glove box (ANX-1; Hirasawa; Japan) with an N2-CO2-H2 atmosphere (85:10:5, v/v/v), and were cultivated using the recommended medium (DSMZ medium 104) and conditions (Ohnishi et al, 2011b).…”

Section: Bacterial Strainsmentioning

confidence: 99%

“…The primer sequence was typical of M. micronuciformis. Mega-X, a primer specific to genus Megasphaera, was used as the reverse primer (Ohnishi et al, 2011b).…”

Section: Primer Designmentioning

confidence: 99%

“…The cells were boiled for 10 min, and the debris was removed by centrifugation at 10,000 g for 10 min at 4°C. Thereafter, 1 µl of the supernatant was used for PCR analysis according to a previous study (Ohnishi et al, 2011b).…”

Section: Dna Extractionmentioning

confidence: 99%

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Study of the distribution of Megasphaera micronuciformis in oral cavities of the Japanese by species-specific polymerase chain reaction (PCR) assay

Ran¹,

Abe²,

Hasegawa³

et al. 2013

Afr. J. Microbiol. Res.

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Megasphaera micronuciformis is an anaerobic microbe isolated from humans. However, since the microbe is strictly anaerobic, its cultivation requires complicated facilities, making detection costly. For rapid, inexpensive detection and identification of M. micronuciformis in the clinical setting, a new technique is necessary. This study aimed to develop a species-specific PCR primer set for the detection of M. micronuciformis. A ribosomal DNA-specific PCR primer Mm2F was designed for M. micronuciformis. Analytical specificity data showed that the PCR primer set Mm2F/Mega-X produced amplicons from M. micronuciformis but not from the other species tested, including 4 Megasphaera species and representative related species. Of the 52 oral samples from Japanese subjects evaluated in our study, 71% were positive for M. micronuciformis, suggesting the likelihood that M. micronuciformis is widely distributed in the oral cavity of the Japanese population.

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“…The universal primer set 20F/1540R was used as the positive control (Ohnishi et al, 2011b). The analytical specificity of the constructed primer set Mm2F/Mega-X was evaluated using PCR.…”

Section: Resultsmentioning

confidence: 99%

Section: Bacterial Strainsmentioning

confidence: 99%

“…The primer sequence was typical of M. micronuciformis. Mega-X, a primer specific to genus Megasphaera, was used as the reverse primer (Ohnishi et al, 2011b).…”

Section: Primer Designmentioning

confidence: 99%

Section: Dna Extractionmentioning

confidence: 99%

See 2 more Smart Citations

Study of the distribution of Megasphaera micronuciformis in oral cavities of the Japanese by species-specific polymerase chain reaction (PCR) assay

Ran¹,

Abe²,

Hasegawa³

et al. 2013

Afr. J. Microbiol. Res.

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show abstract

“…In recent years, various new methods have been adopted in the brewing industry based on cell and microcolony visualisation and extensive analysis of cellular and genetic content 39,41,46 . PCR based methods have been widely evaluated in brewing laboratories in recent years 1,[22][23][24]27,28,34,37,43,49 .…”

Section: Introductionmentioning

confidence: 99%

Occurrence of Pectinatus and Megasphaera in the Major UK Breweries

Paradh

Mitchell

Hill

2011

Journal of the Institute of Brewing

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The occurrence of beer spoilage bacteria belonging to the genera Pectinatus and Megasphaera in ten major UK breweries was investigated. The sampling points were selected from fermentation areas, beer conditioning areas and beer bottling and canning sites. Multiplex PCR methodology was used for detection of three Pectinatus and three Megasphaera species using speciesspecific primers. The presence of six Lactobacillus species was also examined. Overall, 117 samples were analysed from ten breweries; six samples were positive for the presence of Pectinatus species and three samples were positive for the presence of Megasphaera species, while 34 samples were positive for the presence of Lactobacillus species. Lactobacillus species appeared to be the major potential spoilage microorganisms. Although none of the actual beer samples were found to be positive for Pectinatus and Megasphaera species, their occurrence in aerobic brewery environments indicates sanitation problems and revealed the presence of highly established biofilms in some breweries.

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Megasphaera as Lactate-Utilizing Hydrogen-Producing Bacteria

Ohnishi

2015

Microbial Factories

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Development of a 16S rRNA Gene Primer and PCR-Restriction Fragment Length Polymorphism Method for Rapid Detection of Members of the Genus Megasphaera and Species-Level Identification

Cited by 15 publications

References 11 publications

Study of the distribution of Megasphaera micronuciformis in oral cavities of the Japanese by species-specific polymerase chain reaction (PCR) assay

Study of the distribution of Megasphaera micronuciformis in oral cavities of the Japanese by species-specific polymerase chain reaction (PCR) assay

Occurrence of Pectinatus and Megasphaera in the Major UK Breweries

Megasphaera as Lactate-Utilizing Hydrogen-Producing Bacteria

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